The present invention relates to a Leukaemia Inhibitory Factor-Binding Protein (LBP) and more particularly to a soluble LBP, uses thereof and compositions containing same.
Leukaemia Inhibitory Factor (LIF) is a polyfunctional glycoprotein with actions on a broad range of tissue and cell types, including induction of differentiation in a number of myeloid leukaemic cell lines, suppression of differentiation in normal embryonic stem cells, stimulation of proliferation of osteoblasts and DA-1 haemopoietic cells and potentiation of the proliferative action of interleukin-3 (IL-3) on megakaryocyte precursors. Functionally, it is able to switch autonomic nerve signalling from adrenergic to cholinergic mode, stimulate calcium release from bones, stimulate the production of acute phase proteins by hepatocytes and induce loss of fat deposits by inhibiting lipoprotein lipase-mediated lipid transport into adipocytes1.
This array of actions is puzzling since it is difficult to conceive of any situation that would require a coordinated response in all the known target tissues of LIF. Actions of LIF, therefore, are probably designed to be restricted by co-localisation of LIF-producing cells and LIF-responsive cells, with tight regulation of LIF production. However, such an arrangement is likely to result in some release of LIF into the circulatory system including blood and other bodily fluids.
In work leading up to the present invention, the inventors discovered a LIF-binding protein in serum which is capable of inhibiting the biological activity of LIF. The identification of this LIF antagonist will now permit greater control in LIF therapy and to prevent any systemic effects of locally administered LIF which are not therapeutically desirable. It also provides a new agent useful in the treatment of LIF associated diseases or conditions. In a particular embodiment, the inventors have discovered that the inhibitory effect of the LIF-binding protein may be more pronounced in heterologous systems, i.e. where the LIF-binding protein from one mammal is used to inhibit LIF in another mammal.
Accordingly, one aspect of the present invention provides a LIF-binding protein (LBP) in soluble form and isolatable from a mammal.
More particularly, the present invention is directed to an isolated LBP in soluble form and obtainable from a first mammalian species, said LBP capable of inhibiting the ability of LIF from a second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species.
The isolated LBP is preferably biologically pure meaning that it represents at least 20%, preferably at least 50%, even more preferably at least 70% and still more preferably at least 85% of the molecule in a solution or composition as determined by weight, biological activity or other convenient means of measurement. Notwithstanding that the LBP is isolated, it may also be in the form of a composition. According to this aspect of the present invention there is contemplated a composition comprising an LBP in soluble form and obtainable from a first mammalian species, said LBP capable of inhibiting the ability of LIF from a second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species, said composition substantially free of protein molecules not having LBP properties.
The isolated LBP in soluble form and obtainable from the first mammalian species is further characterised in that the LBP has at least a 100 fold higher binding affinity for a LIF from the second mammalian species compared to the binding affinity for a LIF from said first mammalian species.
In accordance with the present invention, the first mammal is preferably a human, mouse or rat or other rodent, pig, cow, sheep or other ruminant, goat, horse or primate. The second mammal may also be a human, mouse or rat or other rodent, pig, cow, sheep or other ruminant, goat, horse or primate. Preferably, the first mammal is a non-human mammal and the second mammal is a human. Most preferably, the first mammal is a mouse and the second mammal is a human.
Accordingly, in a preferred embodiment, the present invention is directed to a LBP in soluble form isolatable from a murine animal. More particularly, the present invention provides a LBP in soluble form isolatable from a murine animal, said LBP capable of greater inhibition of human LIF compared to murine LIF.
The isolated LBP may be the naturally occurring molecule, a naturally occurring derivative, part or fragment thereof or may be a recombinant or synthetic form of the molecule including any recombinant or synthetic derivatives, parts or fragments thereof. The LBP may be naturally glycosylated, partially glycosylated or unglycosylated or may have an altered glycosylation pattern from the naturally occurring molecule. The molecule may, for example, undergo treatment with N-glycanase resulting in a deglycosylated or substantially deglycosylated molecule. A xe2x80x9cderivativexe2x80x9d of LBP is considered herein to generally comprise a single or multiple amino acid insertion, deletion and/or substitution of amino acid residues relative to the naturally occurring sequence or an insertion, deletion and/or substitution of molecules associated with LBP such as carbohydrate moieties. A xe2x80x9cderivativexe2x80x9d is also considered to be a molecule with at least 45% amino acid sequence similarity to the amino acid sequence of the LBP.
Amino acid insertional derivatives of LBP include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical subsitutions are those made in accordance with the following Table 1:
Where the LBP is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis(14) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Maniatis et al(15).
Other examples of recombinant or synthetic mutants and derivatives of the LBP of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the LBP such as carbohydrates, lipids and/or proteins or polypeptides.
In one embodiment, the LBP is truncated at its carboxy terminal end portion to render said LBP soluble.
In another embodiment, the LBP is a fusion molecule between LBPs from said first and second mammalian species.
According to this embodiment, there is provided a fusion polypeptide defining an LBP, said fusion polypeptide comprising first and second amino acid sequences wherein said first amino acid sequence is derivable from an LBP from a first mammalian species and said second amino acid sequence is derivable from an LBP from a second mammalian species wherein the LBP from said first mammalian species is capable of inhibiting the ability of LIF from said second mammalian species to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF from said first mammalian species such that said fusion polypeptide retains the ability to inhibit LIF from said second mammalian species to a greater extent than LIF from said first mammalian species.
In a preferred embodiment, the first mammalian species is a mouse, rat or other rodent, pig, cow, sheep or other ruminant, goat, horse or primate and said second mammalian species is a human. Most preferably, the first mammalian species is a mouse and the fusion polypeptide is referred to as a xe2x80x9chumanisedxe2x80x9d form of the mouse LBP. Such molecules are particularly advantageous in avoiding or reducing possible induction of an antigenic immune response by administering to said first mammal, an LBP from said second mammal.
According to this most preferred aspect of the present invention, there is provided a fusion polypeptide defining an LBP, said fusion polypeptide comprising first and second amino acid sequences wherein said first amino acid sequence is derivable from an LBP from a mouse and said second amino acid sequence is derivable from an LBP from a human wherein said fusion polypeptide is capable of inhibiting the ability of LIF from a human to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit mouse LIF and wherein administration of said fusion polypeptides into a human results in a substantially reduced immune response against said fusion polypeptide compared to the administration to said human of native or recombinant mouse LBP. Conveniently, an xe2x80x9cimmune responsexe2x80x9d is measured by titre of antibodies specific to a molecule and/or involve extent of a cellular immune response.
The fusion polypeptides can be prepared by a range of suitable methods but conveniently is by a method similar to the method employed by the inventors to map the site on the hLIF molecule that confers both binding to the hLIF receptor xcex1-chain and the unusual high affinity binding to the mouse LIF receptor xcex1-chain (mLBP) similar to that described in the experiment summarised in FIG. 9. In particular, a hLBP molecular frame work is used to construct a series of mouse-human mLBP chimaeric molecules in order to determine the minimum number of hLIF amino acid residues that is necessary to substitute into the hLBP sequence in order to create a molecule that has the properties that are peculiar to hLBP.
Reference in the specification and claims herein to xe2x80x9cLBP1xe2x80x9d includes reference to a fusion polypeptide defining an LBP as defined above.
The terms xe2x80x9canaloguesxe2x80x9d and xe2x80x9cderivativesxe2x80x9d also extend to any functional chemical equivalent of the LBP characterised by its increased stability and/or efficacy in vivo or vitro. The terms xe2x80x9canaloguesxe2x80x9d and xe2x80x9cderivativesxe2x80x9d also extend to any amino acid derivative of the LBP as described above.
Analogues of LBP contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the LBP molecule or its analogues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidiation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5xe2x80x2-phosphate followed by reduction with NaBH4.
The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid lidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Other types of modifications include iodination of tyrosine and biotinylation of lysine.
Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group-specific reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides could be conformationally constrained by, for example, incorporation of Cxcex1 and N60-methylamino acids, introduction of double bonds between Cxcex1 and Cxcex2 atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The LBP molecule of the present invention may also be pegolated using polyethylene glycol (PEG) or other equivalent or similar fatty acid to aid in increasing stability and/or the half life of the protein. In this regard, reference can conveniently be made, for example, to U.S. Pat. No. 5,089,261 which describes the use of PEG to increase the in vivo half life of interleukin 1.
Conveniently, the LBP may be in a purified or semi-purified form from blood, serum or other biological fluid sample. More conveniently, the LBP can be purified or semi-purified by sequential fractionation using affinity chromatography on an immobilised LIF column, anion exchange chromatography, size exclusion chromatography and preparative native polyacrylamide gel electrophoresis. One or more of the foregoing steps may be altered or a similar or equivalent step substituted therefor without departing from the scope of the present invention provided the LBP is enriched from a particular biological sample.
The LIF used in the LIF affinity column is generally of first mammalian origin, i.e. same mammalian or gin for both LBP and LIF although any LIF capable of binding the LBP to be purified can be used in the affinity column.
Conveniently, the purification of the LBP is monitored by binding to labelled LIF and preferably radioactively labelled LIF, such as using known 125I-LIF binding assays. Other means of monitoring LBP activity can also be used, such as specific antibody binding or inhibition of LIF activity through competitive assays.
The murine LBP (mLBP) in accordance with the preferred aspects of the present invention when purified as generally described above has an apparant molecular weight as determined on SDS-PAGE of approximately 90,000xc2x110,000 daltons in glycosylated form and 65,000xc2x110,000 daltons in another glycosylated form and specifically binds 125I-murine LIF (mLIF) with an equilibrium dissociation constant of about 0.5-2 nM. Furthermore, mLIF is approximately 1,000-10,000-fold less effective than human LIF (hLIF) in competing with 125I-hLIF for binding to mLBP. More significantly, however, as shown by the in vitro effects herein described, mLBP is at least 100-fold and is generally about 1,000-fold more active as an inhibitor of hLIF than of mLIF. In addition, the direct binding affinity of mLBP for hLIF is approximately 100 times higher than that for mLIF.
In the most preferred embodiment of the present invention, the mLBP has an amino acid sequence in the N-terminal region comprising Gly-Val-Gln-Asp-Leu-Lys-Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro -Leu (SEQ ID No. 1), is soluble, particularly in aqueous buffered solutions and has an apparant molecular weight in the glycosylated form of 90,000xc2x110,000 daltons, and preferably 90,000xc2x15,000 daltons as determined by SDS-PAGE. On the basis of treatment with N-glycanase, one deglycosylated form has a molecular weight of approximately 65,000xc2x115,000 daltons and preferably 65,000xc2x110,000 daltons and another deglycosylated form has a molecular weight of approximately 50,000xc2x110,000 daltons as determined by SDS-PAGE. The present invention extends to LBP molecules having an N-terminal amino acid sequence with at least 45%, preferably at least 55%, more preferably at least 65% and still more preferably at least 75-85% and even more preferably greater than 90% similarity to the amino acid sequence: Gly-Val-Gln-Asp-Leu-Lys-Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu (SEQ ID No. 1).
The present invention extends to nucleic acid molecules and preferably isolated nucleic acid molecules comprising a sequence of nucleotides encoding or complementary to a sequence encoding an LBP as hereinbefore described including a fusion polypeptide defining an LBP. The nucleotide sequence may correspond to the naturally occurring amino acid sequence of the LBP or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto. Preferably, the nucleic acid encodes an LBP with an N-terminal amino acid sequence comprising Gly-Val-Gln-Asp-Leu-Lys Cys-Thr-Thr-Asn-Asn-Met-Arg-Val-Trp-Asp-Cys-Thr-Trp-Pro-Ala-Pro-Leu (SEQ ID No. 1) or contains a nucleotide sequence capable of hybridising under low, preferably under medium and more preferably under high stringency conditions to a nucleotide sequence encoding the above stated amino acid sequence. Put in alternative terms the hybridising nucleotide sequence is at least 45%, preferably at least 55%, more preferably at least 65-75% and even more preferably greater than 85% similar to the nucleotide sequence encoding the above-stated amino acid sequence.
For the purposes of defining the levels of stringency, reference can conveniently be made to Sambrook et al(15) at pages 387-389 where the washing step at paragraph 11 is considered herein to be high stringency. A low stringency wash is defined herein to be 0.1%-0.5% w/v SDS at 37-45xc2x0 C. for 2-3 hours and a medium level of stringency is considered herein to be 0.25%-5% w/v SDS at xe2x89xa745xc2x0 C. for 2-3 hours. The alternative conditions are applicable depending on concentration, purity and source of nucleic acid molecules.
In a particularly preferred embodiment, the nucleic acid molecule of the present invention is in an expression vector capable of replication and expression in eukaryotic organisms (e.g. CHO cells or other mammalian cells, yeast cells, insects cells) and/or in prokaryotic organisms (e.g. E. coli). Such expression vectors and cells transformed with same are convenient sources of the recombinant LBP molecules of the present invention.
The LBP of the present invention will be particularly useful as an inhibitor of the systemic effects of locally produced or administered LIF. Where the use of a heterologous LBP relative to the mammal to be treated is significantly more active than homologous LBP (i.e. LBP from the same species of mammal), then this high activity is particularly advantageous in reducing, for example, the immunological consequences of introducing the heterologous protein into a mammal. The high activity will also enable the administration of as little LBP as possible to ensure that the LBP can be localised to a particular site and cannot disseminate to other areas of the mammal. It may also be important in conjunction with LIF therapy to maintain effective levels of LBP in the circulatory fluids including serum so as to prevent dissemination of LIF administered in the course of the therapy, such as where LIF is locally administered.
Accordingly, another aspect of the present invention contemplates a method of inhibiting the activity of LIF in a mammal comprising administering to said mammal, an effective amount of a soluble LBP.
More particularly, the present invention contemplates a method of inhibiting the activity of LIF in a mammal comprising administering to said mammal, an effective amount of a soluble heterologous LBP wherein said heterologous LBP is capable of greater inhibition of the LIF in the mammal to be treated when compared to a LIF of same mammalian origin to the LBP.
Preferably, the mammal to be treated is a human and the LBP is mLBP.
Preferably, this method is used in the inhibition of the systemic effects of locally produced LIF which are therapeutically undesirable, unintended or unwanted.
Administration may be by any convenient means applicable to the condition being treated but is particularly conveniently administered locally to the site where LIF is to be inhibited. Alternatively, the LBP may be administered to elevate serum levels while LIF is administered locally.
The effective amount of LBP will vary depending on the mammal and condition to be treated but car, range from serum levels of 0.001 xcexcg/ml to 100 xcexcg/ml, preferably 0.01 xcexcg/ml to 50 xcexcg/ml, more preferably 0.1 xcexcg/ml to 20 xcexcg/ml and most preferably 0.5 xcexcg/ml to 10 xcexcg/ml. The amount required will be that amount required to completely or partially inhibit LIF activity or at least reduce it to a clinically acceptable level. Furthermore, the xe2x80x9cLIF activityxe2x80x9d may be all activities associated with LIF or only some of these activities and may be measured in any number of convenient ways such as in a bioassay(9) or a receptor-binding assay.
The LBP may be administered alone or in combination with other active compounds such as, but not limited to, cytokines, antibiotics, anti-cancer agents or immuno-stimulatory or reducing compounds. Administration of the LBP and other active compounds may be by simultaneous or sequential administration. Furthermore, whether the LBP is administered alone or in combination with other compounds, a single dose of LBP may be sufficient or multiple doses or continuous infusion may be required depending on the condition and mammal to be treated and whether any adverse clinical reactions appear.
Yet a further aspect of the present invention provides a pharmaceutical composition comprising LBP as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents. The preparation of pharmaceutical composition is discussed generally in Remington""s Pharmaceutical Sciences, 17th ed. Mach Publishing Co., Easton, Pa., USA. Alternatively, the LBP may be administered genetically using transgenic animal cells or microbial cells.
The active ingredients of the pharmaceutical composition comprising an LBP as herein described are contemplated to exhibit excellent activity when administered in a dosage regimen adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The active compound may be administered in a convenient manner such as by the oral, topical, intravenous, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredient which comprises an LBP may be required to be coated in a material to protect said ingredient from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. In order to administer the vaccine by other than parenteral administration, the LBP may be coated by, or administered with, a material to prevent its inactivation. For example, the LBP may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
The active compound may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thormerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by, for example, the use in the compositions of agents delaying absorption.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the LBP is suitably protected as described above, the molecule may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in the vaccine compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.5 ug and 20 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
As used herein, pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
To reduce the potentially disadvantageous effects of administering a heterologous LBP to a mammal, it may be possible to derivatise or otherwise alter the heterologous LBP to reduce its antigenicity in the mammal to be treated. This can be accomplished by rendering the LBP more like a protein from the mammal to be treated. For example, the LBP can be coupled or masked with a protein or polypeptide or other suitable molecule from the species of mammal to be treated or coupling or fusing the heterologous LBP or parts thereof to the LBP from the target species. In a particularly preferred embodiment, the murine LBP is rendered non-immunogenic in a human and has been derivativatised more like a human molecule.
In yet another aspect of the present invention, the LBP of first mammalian origin is used to detect LIF of second mammalian origin.
In one embodiment, the method contemplated for detecting LIF in a biological sample said method comprising contacting said biological sample with an immobilised LBP from a mammal wherein the LBP is capable of inhibiting the ability of the first mentioned LIF to induce differentiation of M1 myeloid leukaemic cells in vitro to a greater extent when compared to its ability to inhibit LIF of same mammalian origin as LBP and/or wherein said LBP has at least a 100-fold higher binding affinity for said first mentioned LIF compared to the binding affinity of LIF of same mammalian origin as said LBP wherein said contact is for a time and under conditions sufficient for a complex to form between the immobilised LBP and the LIF in the sample; contacting the LBP-LIF complex with an antibody specific for said LIF and labelled with a reporter molecule capable of providing a signal; determining the presence of bound LIF on the basis of the signal produced by said reporter molecule.
In an alternative embodiment, the LBP-LIF complex is contacted with an unlabelled antibody specific to said LIF and then LIF is detected by a second antibody labelled with a reporter molecule and specific to said first antibody.
In yet another alternative embodiment, the LIF in the sample is immobilised (e.g. by an immobilised antibiody) and then bound LIF detected by labelled LBP or first by unlabelled LBP followed by a labelled antibody specific to said LBP.
An embodiment of this aspect of the present invention is described hereinafter with reference to the preferred embodiment of using mLBP to detect hLIF. The present invention, however, is not so limited and extends to the use of LBP and LIF from other mammals.
According to this embodiment, there is contemplated a method of detecting hLIF in a biological sample, said method comprising contacting said sample to mLBP as hereinbefore defined, immobilised to a solid support for a time and under conditions sufficient for a mLBP-hLIF complex to form and then detecting for the presence of said mLBP-hLIF complex.
In a particularly preferred method, the mLBP-hLIF complex is detected by contacting the complex with an antibody for hLIF with the antibody itself being labelled with a reporter molecule or with an additional step of contacting the mLBP-hLIF-antibody complex with a labelled second antibody capable of binding to the first antibody.
The antibodies may be polyclonal or monoclonal and both are obtainable by immunization of a suitable animal with hLIF and either type is utilizable in the LIF assay. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of hLIF, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of LIF assay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; European Journal of Immunology 6: 511-519, 1976).
The presence of a hLIF may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or xe2x80x9csandwichxe2x80x9d assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, mLBP is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an mLBP-hLIF complex, an antibody specific to the hLIF, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated allowing sufficient time for the formation of a complex of mLBP-hLIF-labelled antibody. Any unreacted material is washed away, and the presence of the hLIF is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound mLBP. In accordance with the present invention the sample is one which might contain hLIF and includes biological fluid (e.g. blood, serum, tissue extract) fermentation fluid and supernatant fluid such as from a cell culture.
In the typical forward sandwich assay, mLBP or a hLIF-binding part thereof is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing mLBP to the polymer. After immobilising the mLBP, the polymer-mLBP complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25xc2x0 C.) to allow binding of hLIF in the sample to the immobilised mLBP. Following the incubation period, the complex is washed and optionally dried and then incubated with an antibody specific for hLIF. The antibody is linked to a reporter molecule which is used to indicate the binding of hLIF to mLBP. Alternatively, a second antibody conjugated to a reporter molecule and capable of binding to the first antibody may be used.
An alternative method involves immobilizing the target molecules (i.e. hLIF) in the biological sample and then exposing the immobilized target to mLBP which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling of mLBP. Alternatively, a labelled antibody, specific to mLBP is exposed to the complex to form a tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
By xe2x80x9creporter moleculexe2x80x9d as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the hLIF-specific antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. Generally, the enzyme-labelled antibody is added to the mLBP-hLIF complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate will react with the enzyme linked to the antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. xe2x80x9cReporter moleculexe2x80x9d also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fluorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the mLBP-hLIF. After washing off the unbound reagent, the tertiary complex is then exposed to the light of the appropriate wavelength and the fluorescence observed indicates the presence of the hapten of interest. Immunofluoresence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluiminescent molecules, may also be employed. The above considerations apply where the antibody is an anti-immunoglobulin and is labelled so that a quaternay complex is obtained.
Furthermore, the mLBP of the present invention may be packaged in kit form, for example, to conduct an assay for LIF. The kit is in compartmental form adapted to contain mLBP and may further comprise in the same or different compartments the reagents for the LIF assay.
The foregoing description is equally applicable to the use of LBP from a first mammalian species to detect LIF from a second mammalian species as hereinbefore defined. Furthermore, variations such as binding LIF using immobilised antibodies and then detecting the bound LIF with LBP conjugated to a reporter molecule or using first LBP then LBP-binding antibody conjugated to a reporter molecule.
The present invention is further described by reference to the following non-limiting Figures and/or Example.